M. tuberculosis (Mtb) resistant to multiple drugs (MDR-Mtb) is a bioterrorism threat and global challenge to health. Yet little new chemotherapy against Mtb has emerged in decades. A fresh approach to chemotherapy is to target a pathway in the pathogen that is essential for the pathogen to survive in the host, such as resistance to the oxidative and nitrosative stress imposed by macrophages. This application is based upon preliminary identification of Mtb's proteasome as a previously unrecognized, essential pathway by which Mtb appears to protect itself against oxidative/nitrosative stress. A transposon (tn)-mutagenized library of Mtb containing 10,100 clones (theoretically enough to carry mutations in all 4,000 ORFs) was screened for increased sensitivity to nitrite under conditions thought to mimic the environment in the macrophage phagosome. Thirteen clones (0.013%) were hypersusceptible to nitrite. Five clones carried independent mutations in 2 genes encoding putative components of the proteasome. Actinomycetes such as Mtb are unusual among eubacteria in possessing proteasomes. Among Actinomyctes, Mtb is further unusual in lacking most of other known multimeric, cylindrical, ATPase-dependent bacterial proteases. Thus Mtb may depend on its proteasome to remove irreversibly oxidized, nitrated or nitrosated proteins. This application will test the following hypotheses: (a) The proteasome is essential for survival of Mtb under oxidative/nitrosative stress such as encountered in the mammalian host. (b) Inhibitors can be identified that reproduce the phenotype of disrupting proteasomal genes, thereby making Mtb susceptible to eradication by host defenses. The proposed combination of genetic and chemical targeting, tested in cell-free systems, cultured Mtb, infected macrophages and infected mice, will establish whether the Mtb proteasome represents a new target for anti-infective chemotherapy.